NON LINEAR OPTICAL METHODS IN NEAR FIELD SCANNING

本研究计划将双光子激发（TPE）荧光和二次谐波产生（SHG）的非线性光学方法与近场扫描光学显微镜（NSOM）相结合，研究膜生理学的各个方面。由于可用的膜染色染料的非线性特性增强和远场背景贡献减少，这些方法将提供比现有的线性激发方法更高的对比度，同时保持亚光学分辨率。化学成分和物理性质在细胞功能中很重要，但光学显微镜无法提供必要的分辨率来揭示这些细节。在局部分子水平上，膜电位、弹性或亲水性等特性会发生变化，这些变化可能对细胞功能很重要。NSOM仪器将用于将这些特性与活细胞中的局部化学成分（例如胆固醇与脂质比率）联系起来。荧光寿命测量和随时间变化的荧光各向异性测量将用于探测荧光团周围的局部环境。二次谐波的产生是由诱导极化而不是吸收产生的，潜在的敏感染料将用于产生良好的对比度，而不会产生明显的光漂白。SHG结合极化分析将用于研究膜中荧光团的取向。SHG也将被用作测量膜电位的替代方法。最初的实验将在模型细胞系上进行，例如3T3成纤维细胞和N1E-115未分化和分化的神经母细胞瘤细胞，但一旦投入使用，该设备将成为通用的高分辨率活细胞成像工具。该方案的一个关键方面将是构建腔倾倒钛蓝宝石振荡器，以提供更高的脉冲能量，从而为SHG实验获得高信号电平。

In this research program, the non-linear optical methods of two-photon excitation (TPE) fluorescence and second harmonic generation (SHG) will be combined with near-field scanning optical microscopy (NSOM) to study aspects of membrane physiology. Due to the enhanced non-linear properties of available membrane staining dyes and decreased far-field background contributions, these approaches will provide increased contrast over existing linear excitation methods while maintaining sub- optical resolution. Chemical composition and physical properties are important in cell function but optical microscopy cannot provide the necessary resolution to uncover these details. At he local molecular level, properties such as membrane potential, elasticity, or hydrophilicity, will vary and these variations may be important in cell function. The NSOM apparatus will be used to correlate such properties to local chemical composition, e.g. cholesterol to lipid ratio, in living cells. Fluorescence lifetime measurements and time-dependent fluorescence anisotropy measurements will be used to probe the local environment around fluorophores. Second harmonic generation arises from an induced polarization rather than absorption and potential sensitive dyes will be used to generation good contrast without appreciable photobleaching. SHG combined with polarization analysis will be used study the orientation of fluorophores in membranes. SHG will also be used as an alternative method to measure membrane potential. The initial experiments will be on model cell line, e.g. 3T3 fibroblast and N1E-115 undifferentiated and differentiated neuroblastoma cells but once operational, the apparatus will be general purpose high resolution tool for live cell imaging. A key aspect of this proposal will be constructing cavity dumped titanium sapphire oscillator to provide higher pulse energy so that high signal levels can be obtained for the SHG experiments.